Veterinary Parasitology 203 (2014) 21–28
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Adaptation of a commercial ELISA to determine the IgG avidity in sheep experimentally and naturally infected with Neospora caninum S.S. Syed-Hussain a,b , L. Howe b , W.E. Pomroy b,∗ , D.M. West b , S.L. Smith b , N.B. Williamson b a Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia b Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand
a r t i c l e
i n f o
Article history: Received 1 August 2013 Received in revised form 7 January 2014 Accepted 7 January 2014
Keywords: N. caninum Serology Avidity IgG ELISA Sheep
a b s t r a c t Recent reports indicate Neospora caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the epidemiology of neosporosis in sheep is limited. This study aimed to adapt and validate a commercially available ELISA assay as an IgG avidity assay to discriminate between acute (primary and re-inoculated) and chronic N. caninum infections in sheep. In addition, it was used to compare the antibody avidity values between lambs from ewes inoculated with N. caninum either during the pregnancy or in the previous year. The avidity assay was undertaken by using 6 M urea for the ﬁrst wash after incubation with the primary antibody in the commercial ELISA (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia). Sequential serum samples were obtained from naïve ewes (n = 16) experimentally inoculated with live N. caninum tachyzoites. All ewes were seropositive by two weeks post-inoculation and remained seropositive for 20 weeks post-inoculation. There was a linear relationship between time after inoculation and avidity values (p < 0.05) over the ﬁrst 24 weeks. In Week 4, all animals had avidity values 35%. These results suggest that an avidity value of 35% is indicative of a chronic infection. The assay was then validated using samples from other groups of experimentally inoculated sheep as well as samples from naturally infected ewes. When comparing sample to positive ratio (S/P) and avidity values from lambs born from recently inoculated ewes with those from ewes inoculated the previous year and re-inoculated in the current year, it was possible to differentiate the lambs at 2 weeks of age. Lambs from recently inoculated ewes had low S/P and avidity values at 2 weeks of age which increased by 12 weeks of age. In comparison, lambs from re-inoculated ewes had high S/P and avidity values at 2 weeks of age, due to maternal antibody inﬂuence but values were similar to those from lambs that were born from recently inoculated ewes at 12 weeks of age. Avidity values for four naturally infected ewes were all >60% indicating chronic infection. These results suggest that the assay is able to discriminate between recent and chronic infection in sheep as well as able to differentiate lambs with maternal immunity compared to their own de novo immunity. As such it can be utilized to understand the kinetics of N. caninum infection in sheep. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding author. E-mail address: [email protected]
(W.E. Pomroy). 0304-4017/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2014.01.003
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2. Materials and methods
Neospora caninum is an obligate intracellular apicomplexan protozoan that is recognised as a major cause of abortion in the dairy industry worldwide (Dubey, 2003). The disease can persist in cattle over generations due to the high efﬁciency of vertical transmission (Dubey, 2003). Serology plays an important role in investigating abortion outbreaks and commonly acts as a primary test undertaken to determine the cause or agent involved. Knowing when infection occurs is important for diagnostic purposes. Most serological assays for N. caninum can only determine that the animal has been infected but not when this occurred (Björkman and Uggla, 1999b; Dubey and Schares, 2006). In addition, studies have shown that N. caninum antibody titres may ﬂuctuate after infection for the duration of the animal’s life and that magnitude of the antibody titres does not necessarily provide information on the duration of the infection (Stenlund et al., 1999; Okeoma et al., 2004; Nogareda et al., 2007). Avidity assays have been developed to determine the functional afﬁnity of speciﬁc immunoglobulin G (IgG) antibodies for a range of pathogens including Rubella virus (Hedman and Seppala, 1988), Toxoplasma (Hedman et al., 1989) and cytomegalovirus (Bodeus and Goubau, 1999). Such assays enable the duration since exposure to the pathogens to be estimated in animals and are based on the principle that antibodies produced soon after a primary infection have a low afﬁnity or binding strength towards the antigen as compared to those produced later in a chronic infection or as a B-cell memory response (Jenum et al., 1997). For estimation of IgG avidity, a hydrogen-bond disrupting agent is used, such as urea, that will lead to IgG antibodies with low avidity being dissociated while those with high avidity remain bound to the antigen. On this basis, the avidity of the IgG can be determined to enable discrimination between acute and chronic infection. In 1999, Björkman et al. described an IgG avidity ELISA used to distinguish between acute and chronic N. caninum infections in cattle (Björkman et al., 1999a). Since this initial report, a number of avidity assays for N. caninum infections in cattle have been described and the majority use ELISA technology. A variety of antigen preparations have been used for these including membrane antigens incorporated into iscoms (immunostimulating complexes) (McAllister et al., 2000; Guy et al., 2001; Dijkstra et al., 2002; Björkman et al., 2003, 2005), somatic or whole tachyzoite sonicated lysate (Gottstein et al., 1998; Maley et al., 2001; Sager et al., 2003; Aguado-Martinez et al., 2005) and afﬁnity puriﬁed surface antigen (Schares et al., 2002). The aim of the present study was to adapt and validate a commercially available ELISA assay (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia) as an IgG avidity assay for N. caninum infection in sheep. The study was conducted using sequential serum samples obtained from previously naïve sheep experimentally inoculated with live N. caninum tachyzoites. The results were subsequently validated with serum samples obtained from other experimentally and naturally infected sheep. Comparison of avidity values of lambs born from newly or re-inoculated dams with N. caninum was also undertaken.
2.1. Experimental design This trial consisted of three parts. The ﬁrst was a challenge study to determine the development of sample to positive ratio (S/P) and IgG avidity values of ewes experimentally inoculated with N. caninum tachyzoites. The second was to validate the IgG avidity assay by comparing the avidity values of serum samples between those obtained in the challenge study and from other experimentally and naturally infected sheep. The third was to compare the S/P and avidity values of lambs born from ewes experimentally inoculated with N. caninum. Details of the experimental groups are shown in Table 1.
2.1.1. Sequential serum samples from experimentally inoculated sheep used to develop the avidity assay Serum samples were collected from 16 ewes (Group 1) that were experimentally inoculated with 106 live N. caninum (NcNZ1) tachyzoites intravenously two months prior to natural mating in Year 1. The inoculums used were prepared as described previously (Syed-Hussain et al., 2013). In Year 2 of the study, the 16 ewes were mated again from Week 56 post initial inoculation for two oestrus cycles. At Week 72 (Day 120 of gestation in Year 2), 9 ewes in Group 1 were re-inoculated with 107 live N. caninum tachzyoites (now known as Group 3 from Week 72) while the other 7 were not re-inoculated and remained as Group 1. Five additional ewes were used as negative controls (Group 2) and were kept together with the inoculated animals to rule out the occurrence of horizontal transmission of N. caninum in the farm throughout the study. Blood samples were collected at Weeks 0, 2, 4, 8, 12, 16, 20, 24, 72, 76 and 84 post inoculation.
2.1.2. Serum samples from other experimentally and naturally infected sheep to validate the avidity assay Samples were obtained from other studies where sheep had been experimentally inoculated and also from sheep naturally infected with N. caninum. Details of the these additional serum samples are: Group 4–8 rams (age 6 months) 2 weeks after inoculation with 107 live N. caninum tachyzoites as previously described by Syed-Hussain et al. (2013); Group 5–6 mixed age ewes, 4 weeks post inoculation with 107 live N. caninum tachyzoites; Group 6–15 lambs with a mean age of 12 weeks born from naïve ewes in Group 5 that were experimentally inoculated with 107 live N. caninum tachyzoites on Day 120 of gestation in Year 2 (mean time post inoculation of 17 weeks); Group 7a–5 lambs which were seropositive at 12 weeks of age that were born from ewes in Group 3 after re-inoculation in Year 2 (mean time post inoculation for these lambs is also 17 weeks); Serologically positive ewes, naturally infected at an unknown time with N. caninum and included Unknown (UK) 1 (tested pre mating, during pregnancy and after an abortion), UK 2 tested soon after abortion at mid pregnancy, UK 3 and UK 4 both sampled immediately pre mating.
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Table 1 Descriptions of experimentally and naturally infected sheep with N. caninum used in the Ig G avidity assay. Groups
Number of animals
Group 2 Group 3
Group 4 Group 5 Group 6
8 6 15
Group 7b UK
Ewes, inoculated with 106 live N. caninum tachyzoites intravenously two months prior to mating in Year 1 Control ewes not inoculated Ewes in Group 1, re-inoculated with 107 live N. caninum tachzyoites at Day 120 of gestation in Year 2 Rams, 2 weeks after inoculation with 107 live N. caninum tachyzoites Naïve ewes, 4 weeks post inoculation with 107 live N. caninum tachyzoites in Year 2 Lambs, 12 weeks of age that were born from naïve ewes in Group 5 that were experimentally inoculated with 107 live N. caninum tachyzoites on Day 120 of gestation in Year 2 Lambs, 12 weeks of age that were born from ewes in Group 3 that were seropositive by ELISA at 2 and 12 weeks of age Lambs born from Group 3 ewes that were only ELISA seropositive at 2 weeks of age Naturally infected ewes
2.1.3. Comparison of S/P and avidity values of lambs Only sera from lambs that were seropositive at both 2 and 12 weeks of age that were born to ewes that were either inoculated in Year 1 or inoculated in Year 1 and Year 2 were selected and compared. For the former, this included lambs from Group 6 (n = 4) and for the latter this included lambs in Group 7a (n = 5) together with the other lambs born from Group 3 ewes that were only ELISA seropositive at 2 weeks of age (Group 7b; n = 7).
for samples either with or without the urea wash and expressed as a percentage as shown below. For Group 1, avidity values were calculated for all samples from Week 2 onwards while for other groups only seropositive samples (S/P value ≥ 11.8%) were tested. Avidity Index % =
OD (urea treated samples) × 100 OD (untreated)
2.4. Western blot 2.2. IgG ELISA All sera collected during this trial were tested using a commercial ELISA assay (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia) and all the assay kits used in this study were from the same batch. This indirect assay utilises N. caninum antigen with anti-ruminant IgG conjugates and detects antibodies against N. caninum antibodies in samples from cattle, goats and sheep. Results were calculated as a corrected sample to positive ratio (S/P) and the value was expressed as a percentage. According to the manufacturer’s instructions a value greater than 30% is considered as suspect and above 40% as positive for N. caninum. However in this study, the ﬁndings of Reichel et al. (2008) were adopted where an S/P value of ≥11.8% was regarded as positive. 2.3. IgG avidity ELISA The IgG avidity ELISA was developed utilising the same commercial assay as in Section 2.2. Brieﬂy, samples were assayed in duplicates on the same plate. The protocol was the same except for a modiﬁcation of the ﬁrst wash after incubation with the primary antibody. For one series, the ﬁrst wash was with 200 l 6 M urea (Gibco BRL, Life Technologies, NY, USA) diluted in the wash provided by the manufacturer. For the second series, washing was with 200 l of the provided solution. This ﬁrst wash for both series was conducted with mild agitation for 5 min at 37 ◦ C. The plate was then further washed twice without urea as per the manufacturer’s instructions. Positive and negative controls provided by the manufacturer were also included in both duplicates. The avidity index was calculated by comparing the optical density (OD) ratios
At least one serum sample from each animal was analysed using a western blot to conﬁrm they were seropositive using the technique as described previously (Syed-Hussain et al., 2013). 2.5. Statistical analysis S/P values for the commercial ELISA and the subsequent avidity values were found to be normally distributed. General linear model repeated measures with GreenhouseGeisser correction were performed and post hoc tests using the Bonferroni method were undertaken to determine and compare the differences of mean S/P and avidity values between sampling times in Group 1. A one way ANOVA was used to compare the avidity values between the other groups. The data spanning Week 2 to Week 24 for Group 1 was also described with a regression line. Statistical analyses were performed using SPSS (Version 20, IBM® , SPSS® Statistic) and all statistical analyses were considered signiﬁcant when p < 0.05. 3. Results 3.1. Challenge study: sequential serum samples from experimentally inoculated sheep used to develop the avidity assay 3.1.1. IgG ELISA A summary of the S/P values is shown in Fig. 1. All inoculated ewes in Group 1 were positive by western blot at 2 weeks post inoculation. In Group 1, all 16 ewes had seronegative ELISA S/P values in Week 0 which then increased to a peak in Week 4 followed by a gradual decline
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Fig. 1. Box and whisker plot of S/P values of ewes in Group 1 ( ; Week 0–72, n = 16; Week 76 and 84, n = 7) which were inoculated with N. caninum in Week 0. Group 2 are un-inoculated control ewes (; n = 5). Group 3 (; n = 9) represent ewes from Group 1 that were re-inoculated in week 72 (arrow indicates day 120 gestation). The mean value () and outliers () are as indicated. Means that do not share a similar letter are signiﬁcantly different (p < 0.05).
until Week 84. All ewes were serologically ELISA positive by Week 2 until Week 20 but from Week 24 onwards there were some ewes on each sampling occasion that were serologically negative. Two ewes were seronegative (S/P = 10.3% and 11.5%) in Week 24 but were seropositive on all subsequent occasions. One ewe became seronegative from Week 72 onwards with S/P values ranging from 4.0% to 8.1%. In addition, in Week 76, 3 of 7 ewes were seronegative but all were found to be seropositive again in Week 84. The upper outlier shown in Week 12, 20 and 24 was the same ewe which had consistently high S/P values throughout the study. The mean S/P values overall differed signiﬁcantly between time points (F (2.95, 44.31) = 27.803, p < 0.001). Post hoc comparisons between weeks are also shown in Fig. 1. In Week 2 post inoculation, mean S/P values were 26% and were signiﬁcantly higher than Week 0 (p < 0.05). S/P values in Week 4 (p < 0.05) were signiﬁcantly higher than in Week 2 but no signiﬁcant differences were found between the S/P values from Week 4 until Week 16 (p > 0.05). The mean S/P values in Week 20 to Week 84 were not signiﬁcantly different from each other (p > 0.05) but were signiﬁcantly lower than the values from Week 4 to Week 16 (p < 0.05). Un-inoculated ewes in Group 2 (control) had S/P values lower than 11.8% and were all serologically negative in Week 0 and in Week 84. They were also negative on western blot at Week 84. In Group 3, after re-inoculation of ewes (n = 9) at Week 72, their mean S/P values increased to 49% and then 59% in Week 76 and Week 84 respectively as compared to those that were not re-inoculated in Group 1 (n = 7) which remained low with a mean S/P value of 17% and 19% respectively in these same two weeks. There was a signiﬁcant difference in mean S/P values between Group 1 and 3 (p < 0.05) in Week 76 and Week 84.
3.1.2. IgG avidity assay of Group 1 The development of IgG avidity values for Group 1 was able to be determined from Week 2 post inoculation and is
shown in Fig. 2. The regression line from Week 2 to Week 24 is described below in Eq. (1) and also shown in Fig. 2 together with a 95% prediction interval. Avidity % = 24.47 + 1.032 WEEKS PI(p < 0.05; R2 = 54%) (1) Mean avidity values increased gradually and when analysed with repeated measures they differed signiﬁcantly between time points (F (1, 110) = 130.86, p < 0.001) as shown in Fig. 2. In Week 2 the mean avidity values were not signiﬁcantly different (p > 0.05) from those in Week 4 but the values in Week 8 were signiﬁcantly higher than those in Week 2 and 4 (p < 0.05). In Weeks 2 and 4, all but 2 animals had avidity values 0.05). By Week 24, more than half of the group had avidity values of ≥45%. Subsequently all animals in Weeks 72, 76 and 84 had avidity values of ≥45%. In general, from Week 72 the mean avidity values plateaued for the remainder of the study. 3.2. Validation of IgG avidity assay: serum samples from other experimentally inoculated and naturally infected sheep compared to Group 1 Fig. 2 shows the avidity values for other groups compared to the values from Group 1. In Group 3, one month after re-inoculation (Week 76), no signiﬁcant differences of avidity were detected between ewes in Group 1 and 3, although the mean avidity values for Group 1 were signiﬁcantly lower than those of Group 3 (p < 0.05) in Week 84. Rams sampled two weeks post inoculation (Group 4) had a low mean avidity value of 23% (95% prediction interval; 11-41%) while ewes sampled 4 weeks post inoculation (Group 5) developed a mean avidity value of 27% (95%
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Fig. 2. Box and whisker plot of mean avidity values of ewes in Group 1( ; n = 16), Group 3 (n = 9; ewes from Group 1 re-inoculated in Week 72), Group 4 (n = 7; rams bled at 2 weeks post inoculation), Group 5 (n = 6; ewes bled 4 weeks post inoculation), Group 6 (n = 15; lambs at 12 weeks old from ewes inoculated at Day 120 of gestation), Group 7a (n = 5; lambs at 12 weeks old from ewes re-inoculated at Day 120 of gestation) and values are also shown for four naturally infected ewes where the time of infection is unknown. Circle in the centre () of the bar indicates the mean value and the triangle sign () indicates outliers. Means that do not share a similar letter are signiﬁcantly different (p < 0.05). The regression line from Week 2 to Week 24 is shown (—) with 95% prediction intervals (—).
prediction interval; 14-43%). Avidity values for both these groups fell within the 95% prediction interval with avidity values consistent with recent infection of less than 8 weeks prior. Mean avidity values for 12 week old lambs in Groups 6 (lambs from ewes inoculated once) and 7a (lambs from reinoculated ewes) were 48% and 54% respectively and were not different (p > 0.05) from each other. As these ewes were inoculated on Day 120 of gestation, these lambs presumably had been exposed for 16 to 17 weeks prior to the time of sampling. When their mean avidity values were compared to the prediction intervals for 17 weeks, the values of all but 1 lamb in Group 7a (which had a higher avidity value) fell within the 95% prediction interval (Fig. 2) and are consistent with an infection of greater than 12 weeks prior. Avidity proﬁles for four naturally infected ewes with N. caninum are also shown in Fig. 2. UK1, bled on 3 occasions, consistently had high S/P and avidity values with the latter varying from 80-100%. UK2 was determined to have recently aborted at mid pregnancy and also had a high S/P of 100% with an avidity value of 72%. Both UK3 and UK4 were sampled pre-mating and had S/P values of 12% and 37% respectively with avidity values of 90% each.
2 and 12 weeks of age, whilst the values for Group 7a were signiﬁcantly reduced (p < 0.05) by 12 weeks of age. All lambs in Groups 6, 7a and 7b were positive by western blot when tested at 12 weeks of age.
4. Discussion In the present study, a commercially available IgG ELISA test kit was adapted as an avidity assay to discriminate between acute and chronic N. caninum infection in sheep. The usefulness of this test was determined using sequential sera obtained from sheep experimentally inoculated with N. caninum tachyzoites and validated with sera obtained from other sheep either naturally infected or experimentally inoculated.
3.3. Comparison of S/P and avidity values of lambs born from experimentally inoculated ewes Fig. 3 shows the S/P and avidity values for lambs in Group 7a and those that were seropositive at both 2 and 12 weeks of age in Group 6 as well as values for Group 7b at 2 weeks of age. At 2 weeks of age, S/P and avidity values for lambs in Group 7a were signiﬁcantly higher (p < 0.05) than those in Groups 6 and 7b. Avidity values for Groups 7a and 7b at 2 weeks of age were all >60% and were signiﬁcantly higher (p < 0.05) than those in Group 6. However, at 12 weeks of age, no signiﬁcant difference (p > 0.05) in S/P or avidity values was found between Groups 6 and 7a. No avidity values are available for Group 7b as they were all ELISA seronegative at 12 weeks of age. Avidity values for Group 6 were signiﬁcantly increased (p < 0.05) between
Fig. 3. Box and whisker plot for mean S/P and avidity values at 2 and 12 weeks of age for lambs in Group 6 (; n = 4), Group 7a ( ;n = 5) and. Group 7b (, n = 7). Group 7b were only seropositive at 2 weeks but seronegative at 12 weeks and not subjected to the avidity assay at 12 weeks. Means that do not share a similar letter are signiﬁcantly different (p < 0.05). Circle in the centre () of the bar indicates the mean value.
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Using the ELISA, positive antibody titres were detected by 2 weeks post inoculation and this ﬁnding is consistent with other studies in sheep and cattle (Björkman et al., 1999a; Maley et al., 2001; Buxton et al., 2001; Sager et al., 2003). The speciﬁc IgG levels peaked at Week 4 post inoculation and then gradually declined throughout the study. This trend, with a peak titre one month post inoculation followed by a gradual decrease to 6 months post inoculation, correlates well to the ﬁndings of earlier infection studies in sheep by Buxton et al. (1997, 2001). The S/P values at 28 days are similar to those reported by Weston et al. (2009) using the same isolates and a similar infection protocol. However, the S/P values in these inoculated ewes were lower than those reported by Reichel et al. (2008) but that may be due to an effect of a different isolate and/or breed of sheep. In the current study, the antibody responses increased in ewes re-inoculated in Week 72 and were greater in magnitude compared to those that were left un-inoculated. This is similar to the trend observed in sheep re-infected on Day 90 of gestation (Buxton et al., 2001). In the present study, some animals showed a ﬂuctuating response, with some being considered seronegative especially beyond 6 months after inoculation. Fluctuating antibody titres which become seronegative in previously seropositive animals have been observed in experimentally infected calves (Maley et al., 2001) and especially in pregnant cows (Conrad et al., 1993; Okeoma et al., 2004). This ﬁnding emphasizes the obstacles to making a diagnosis using serology, especially in animals that were chronically infected with N. caninum, as it had been shown that being seronegative does not necessarily mean that the animal was never infected (Howe et al., 2012). It is also worth noting that the ﬁndings in this study suggest that infecting sheep with the NcNZ1 isolate of N. caninum originally isolated from cattle leads to persistent infection as most of the experimentally infected animals’ antibody titres remained elevated above the seropositive cut-off value for more than 20 months post inoculation. As rapid development of antibody titres were detected, it was possible to determine avidity values from 2 weeks post inoculation. A consistent increase in the avidity values over time was detected from Week 2, especially over the ﬁrst 24 weeks and by Week 72 these values had effectively reached a plateau. While the linear relationship between time after inoculation and avidity is signiﬁcant (p < 0.05) over the ﬁrst 24 weeks, the R2 value was only 54% indicating variation in the development of high avidity values between animals. For the ewes experimentally inoculated for the ﬁrst time (Group 1), higher values than expected were recorded at Week 12 which was inconsistent with the trend for other weeks where the mean value was generally near the regression line. The 95% prediction interval, spanning from Week 2 to 24 was quite wide suggesting that individual sampling might have a high variability but the assay would be more useful to assess the avidity of a ﬂock. Investigation of the avidity values in the current study has suggested that an avidity value of 35% is indicative of a more long standing infection. The lower 95% prediction interval (Fig. 2) was 35% at 24 weeks postinfection for Group 1 whilst the regression line intersected
this value much earlier at 8 weeks. For ewes in Group 1, avidity values were 45% for ewes inoculated for >6 months are also in agreement with those from experimentally infected cattle (Björkman et al., 1999a; Sager et al., 2003). Interestingly, although there are some differences, the comparison of avidity values between different assays using different techniques and antigens do not have a marked effect on the extent of the resulting avidity values. In studies where whole antigen lysate of N. caninum was used, it was suggested that cut points of 35% were indicative of low, intermediate and high avidity values respectively (Sager et al., 2003; Aguado-Martinez et al., 2005). However, there were also variations in other aspects of the assay between this and other studies including types of urea, time of urea incubation and number of urea washes used (Hedman et al., 1989; Marcolino et al., 2000; Björkman et al., 2006). The observation made after Week 72 on ewes in Groups 1 and 3, suggested that the combination of both S/P and avidity values could be useful to determine if chronically infected animals were experiencing recrudescence of an infection. Ewes which were not re-inoculated had low S/P values and a high avidity whilst those that were re-inoculated had much higher S/P (p < 0.05) as well as high avidity values. However, to what extent this would be seen in natural infections in animals with oocysts remains to be determined, although similar trends have been observed in cattle when comparing results from
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experiments with infections with tachyzoites to those with oocysts (Björkman et al., 1999a, 2005; Sager et al., 2003). The avidity assay alone was not able to directly differentiate between those that were only inoculated in Year 1 (Group 1) and those that were re-inoculated (Group 3) as seen at Week 76 and 84. This limitation was also observed in other studies (Björkman et al., 2003; Sager et al., 2003; Aguado-Martinez et al., 2005). In the current study, in ewes where the antibody titres ﬂuctuated to below the cut-off point (Fig. 1; Group 1), the avidity values remained high thus making it a potentially useful diagnostic test with these low titre animals. This was also similar to the ﬁndings of other studies using the same method for determining avidity (Holliman et al., 1994; Maley et al., 2001). In ﬁeld situations however, only samples with positive S/P values should be submitted to an avidity test. Unless other diagnostic methods such as western blots are used concurrently, interpreting test results for an individual animal with low antibody titres should be done cautiously as such low antibody titres could be due to non speciﬁc binding and lead to false positive results. At 12 weeks of age, the avidity values for lambs in Groups 6 and 7a (Fig. 2) ranged from 37% to 62%, which is within the 95% prediction interval for Week 20 to 24 onwards. This suggests that the lambs were chronically infected beyond their age, thus indicating congenital transmission. These lambs were born from naïve dams that were either inoculated on Day 120 of gestation (Group 6) or dams that were re-inoculated (Group 7). Assuming the foetal immune system responded from the time of inoculation, these samples at 12 weeks of age would have been equivalent to 17 weeks post exposure. Although they generally fall within the 95% prediction interval, for the 17 week time point (Fig. 2) there are some values that were slightly higher. Though pre-colostral blood was not collected, these lambs were kept as a mob together with control animals that did not seroconvert to N. caninum and these control animals were used to rule out the occurrence of postnatal infection that could have occurred on the farm. These control animals and their lambs remained seronegative throughout the study. Comparison of S/P together with the avidity values can potentially enable differentiation between lambs born from recently inoculated (Group 6) and re-inoculated (Group 7) dams (Fig. 3). At 2 weeks of age, a marked difference (p < 0.05) was seen between these lambs in Group 6 and Group 7a. Lambs from Group 6 had low mean S/P (12%) and avidity (20%) values while Group 7a had high mean S/P (60%) and avidity values (80%). In contrast, at 12 weeks of age, there were no signiﬁcant differences (p > 0.05) in the mean S/P and avidity values between both groups. At 2 weeks of age, antibody responses would be a mixture of both maternal and those derived from the lambs themselves. The half-life of maternal IgG in lambs has been reported to vary from 14 to 25 days (Pearson and Brandon, 1976; Campbell et al., 1977; Whitelaw and Jordt, 1985; Watson, 1992). Thus by 12 weeks of age, the S/P and avidity values would overwhelmingly reﬂect the lamb’s own responses. These values indicate these lambs had mounted their own humoral response. The seven lambs in Group 7b were positive by ELISA at 2 weeks of age with high avidity
values but by 12 weeks of age their S/P values were negative indicating the antibodies at 2 weeks of age were most likely of maternal origin and had waned to being undetectable with the ELISA by 12 weeks. Interestingly all 7 of these lambs were serologically positive by western blot at 12 weeks of age and may suggest that the western blot is sufﬁciently sensitive to detect low levels of the lambs own humoral response. As to whether this would also indicate the detection of low levels of maternal antibodies is unknown as no study on the duration of maternal antibodies of N. caninum in sheep has been reported. High levels of antibodies have been detected at 3-4 months of age in lambs congenitally infected with T. gondii (Dubey and Kirkbride, 1989) and these were considered to be due to an active infection as maternal antibodies will have disappeared by 3 months of age (Dubey, 2010). In this study, it shows that at 2 weeks of age, the avidity assay could be used as a tool to determine if seropositive lambs were born from dams that were recently infected with N. caninum or if they were born from chronically infected dams experiencing recrudescence. This study provides no information on lambs born from chronically infected ewes that were not re-inoculated. In the current study, naturally infected animals had high avidity values indicating they were chronically infected when sampled. Interestingly, these ewes were the only individuals that tested seropositive for N. caninum from their particular mobs. The relevance of these infections could not be determined. The results from the present study have shown this avidity assay was not only easy to use but was also able to discriminate between recent and chronic infection and could provide a useful method for determining the involvement of N. caninum in abortion cases on sheep farms. Although care and critical evaluation are required when examining samples from a single individual as compared to those in a mob, obtaining serial samples over time or from a group of animals would further improve the estimation of the exposure time to the infection. This study has demonstrated that this avidity assay can be utilized to further understand the kinetics of this disease in sheep but it is essential to further evaluate its performance, especially in naturally infected sheep. Acknowledgements Scholarship support for the ﬁrst author was provided by The Ministry of Higher Education, Malaysia and Universiti Putra Malaysia. The authors wish to thank Anne Moss, Barbara Adlington and Elizabeth Burrows for their technical assistance. Financial support for this project was generously provided by the C. Alma Baker Trust. Assistance with purchasing the Chekit* Neospora antibody test kit was provided by IDEXX Laboratories, Australia. References Aguado-Martinez, A., Alvarez-Garcia, G., Arnaiz-Seco, I., Innes, E., OrtegaMora, L.M., 2005. Use of avidity enzyme-linked immunosorbent assay and avidity western blot to discriminate between acute and chronic Neospora caninum infection in cattle. J. Vet. Diagn. Invest. 17, 442–450.
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